PROJECT SUMMARY Multiple sclerosis (MS) patients are three to six times more likely to develop epilepsy compared to the rest of the population. However, while this groups suffers greater morbidity, the pathophysiology of MS-associated seizures is unknown. Our long-term goal is to identify mechanisms linking demyelination to neuronal hyperexcitability and neurodegeneration. The objective in this application is to define the processes by which demyelination itself causes cellular, molecular and circuit changes increasing neuronal excitability. Our central hypothesis is that demyelination is coupled to elevated excitability, loss of parvalbumin (PV)+ interneurons, and dysfunction of astrocyte metabolism/transport. This hypothesis is based on our recently published work demonstrating marked changes in electroencephalography (EEG) and spontaneous seizures in mice fed 0.2% cuprizone diet (CPZ) over a period of 9-12 weeks; and subsequent immunohistochemistry revealed loss of PV+ neurons in the hippocampal CA1 subregion together with widespread gliosis and changes in astrocytic aquaporin-4 (AQP4) expression com- pared to mice on a normal diet. The rationale for the proposed research is that detailed spatiotemporal moni- toring of EEG activity with multielectrode arrays (MEA) in CPZ-treated mice will allow identification of the locus and timing of seizure initiation during chronic demyelination, and this will direct the probe of excitatory/inhib- itory neurotransmission and cellular/molecular changes by immunohistochemical and electrophysiological tech- niques. Based on new preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Define the spatial and temporal generation of chronic demyelination-associated seizures; 2) Evaluate the role of GABAergic neurons with an emphasis of PV neurons in the generation of chronic demyelination-induced sei- zures; 3) Evaluate the role of astrocytes in regional seizure susceptibility during chronic demyelination. Novel electrophysiological and transgenic approaches together with direct comparison to human tissue from patients with MS with and without seizures will elucidate demyelination-associated cellular and molecular changes that lead to seizure susceptibility. The proposed research is significant, because it will advance fundamental knowledge of glial-neuronal interactions in the brain while providing new and rational strategies and treatments for prevention and treatment of MS-associated seizures.